1. Field of the Invention
The present invention generally relates to compositions and methods for differentiating and culturing pluripotent stem cells, the cells created by these methods and their uses thereof.
2. Background Art
Embryonic Stem (ES) cells represent a powerful model system for the investigation of mechanisms underlying pluripotent cell biology and differentiation within the early embryo, as well as providing opportunities for genetic manipulation of mammals and resultant commercial, medical and agricultural applications. Furthermore, appropriate proliferation and differentiation of ES cells can be used to generate an unlimited source of cells suited to transplantation for treatment of diseases that result from cell damage or dysfunction. Other pluripotent cells and cell lines including early primitive ectoderm-like (EPL) cells as described in International Patent Application WO 99/53021, in vivo or in vitro derived ICM/epiblast, in vivo or in vitro derived primitive ectoderm, primordial germ cells (EG cells), teratocarcinoma cells (EC cells), and pluripotent cells derived by dedifferentiation or by nuclear transfer will share some or all of these properties and applications.
The successful isolation, long-term clonal maintenance, genetic manipulation and germ-line transmission of pluripotent cells has generally been difficult and the reasons for this are unknown. International Patent Application WO 97/32033 and U.S. Pat. No. 5,453,357 describe pluripotent cells including cells from species other than rodents. Human ES cells have been described in International Patent Application WO 00/27995, and in U.S. Pat. No. 6,200,806, and human EG cells have been described in International Patent Application WO 98/43679.
The ability to tightly control differentiation or form homogeneous populations of partially differentiated or terminally differentiated cells by differentiation in vitro of pluripotent cells has proved problematic. Current approaches can involve the formation of embryoid bodies from pluripotent cells in a manner that is not controlled and does not result in homogeneous populations. Mixed cell populations such as those in embryoid bodies of this type are generally unlikely to be suitable for therapeutic or commercial use.
The biochemical mechanisms regulating ES cell pluripotency and differentiation are very poorly understood. However, the limited empirical data available (and much anecdotal evidence) suggests that the continued maintenance of pluripotent ES cells under in vitro culture conditions is dependent upon the presence of cytokines and growth factors present in the extracellular serum milieu. A number of such factors such as insulin, IGF(s) and FGF(s) have been found to activate intracellular signaling events through the lipid kinase phosphatidylinositol 3-kinase (PI3-kinase) (Carpenter & Cantley, (1996) Curr. Opin. Cell. Biol., 8: 153-158). In response to the binding of these soluble factors to specific cell surface receptors, PI3-kinase is recruited to the intracellular membrane surface where it initiates a cascade of secondary signaling events leading to the functional regulation of several downstream intracellular targets that influence diverse biological processes. Amongst the downstream targets of PI3-kinase is the protein kinase called ‘mammalian Target Of Rapamycin’ (mTOR). Stimulation of mTOR both precedes and is necessary for activation of ribosomal p70 S6 kinase, a serine/threonine kinase that is pivotal to the regulation of the protein synthetic machinery (Chung et al., (1994) Nature, 370: 71-75).
There is a need, therefore, to identify methods and compositions for the production of a population of cells enriched in a cell lineage through the manipulation of the PI3-kinase signaling pathway, the maintenance or stabilization, and proliferation of these cells, and the products of their further differentiation.